Mitochondria play a central role in mediating apoptosis in a number of apoptotic models (Kroemer et al., Immunol. Today 18:44–51 (1997); Zamzami et al., J. Exp. Med. 183:1533–44 (1996); Zamzami et al., J. Exp. Med. 182:367–77 (1995)). Cells induced to undergo apoptosis show an early disruption of mitochondrial transmembrane potential (ΔΨm) preceding other changes of apoptosis, such as nuclear fragmentation and exposure of phosphatidylserine on the outer plasma membrane. Isolated mitochondria or released mitochondrial products induce nuclear apoptosis in a cell-free reconstituted system (Liu et al., Cell 86:147–57 (1996); Newmeyer et al., Cell 79:353–64 (1994)).
Previous experiments indicated that the pre-apoptotic ΔΨm loss involves the opening of mitochondrial permeability transition (PT) pores, which are high-conductance channels at the inner mitochondrial membrane corresponding to mitochondrial megachannels identified by electrophysiological studies (Kroemer et al., supra; Zamzami et al. (1996), supra; Bernardi et al., Biochim. et Biophys. Acta 1275:5–9 (1996); Zoratti et al., Biochim. et Biophys. Acta 1241:139–76 (1995); Petit et al., FEBS Letters 396:7–13 (1996)). In fact, induction of PT is sufficient to provoke the full spectrum of apoptosis-associated changes. Conversely, agents that prevent opening of PT pores, such as bongkrekic acid, attenuate apoptosis (Kroemer et al., Immunol. Today 18:44–51 (1997); Zamzami et al., J. Exp. Med. 183:1533–44 (1996); Zamzami et al., FEBS Letters 384:53–57 (1996)).
Members of the evolutionarily conserved Bcl-2 family are important regulators of apoptotic cell death and survival. The proteins Bcl-2, Bcl-xL, Bcl-w, A1 and Mcl-1 are death antagonists while Bax, Bak, Bad, Bcl-xs, Bid, and Bik are death agonists (Kroemer et al., Nature Med. 6:614–20 (1997)). Bcl-2 family member proteins are predominantly localized in the outer mitochondrial membrane, but are also found in the nuclear membrane and endoplasmic reticulum (Kroemer et al., supra).
Among Bcl-2 family member proteins, there are several conserved amino acid motifs, BH1–BH4. The pro-apoptotic members of the family, Bax and Bad, contain a BH3 domain that is sufficient to induce cell death (Chittenden et al., EMBO J. 14:5589–96 (1995); Hunter et al., J. Biol. Chem. 271:8521–24 (1996)). Interestingly, the BH3 domain is conserved in the anti-apoptotic proteins Bcl-2 and Bcl-xL. Recently, it was reported that cleavage of Bcl-xL and Bcl-2 in the loop domain removes the N-terminal BH4 domain and converts Bcl-xL and Bcl-2 into a potent pro-death molecule (Cheng et al., Science 278:1966–68 (1997); Clem et al., Proc. Nat. Acad. Sci. USA 95:554–59 (1998)).
NMR structure analysis of a complex between Bcl-xL and a 16 residue peptide encompassing the Bak BH3 domain demonstrated that the BH3 peptide, in an amphipathic alpha-helical configuration, binds with high affinity to the hydrophobic pocket created by the BH1, BH2 and BH3 domains of Bcl-xL (Sattler et al., Science 275:983–86 (1997)). Leucine at position 1 of the BH3 domain core and aspartic acid at position 6 are believed to be critical residues for both heterodimerization and apoptosis induction. In further support of this conclusion, a number of “BH3 only” death promoters have been identified which have no similarity to Bcl-2 beyond their BH3 domain homology (Kelekar et al., Trends Cell Biol. 8:324–30 (1998)). These include Bik, Bim, Hrk, Bad, Blk, and Bid, which cannot homodimerize, but rely on binding to anti-apoptotic proteins such as Bcl-2 to induce cell death.
The exact mechanisms by which Bcl-2 prevents apoptosis remain elusive. In light of the importance of mitochondria in apoptosis and the mitochondrial location of Bcl-2, it appears that one major site where Bcl-2 interrupts apoptotic signals is at the level of mitochondria. It has been shown that Bcl-2 inhibits apoptosis by preventing mitochondrial permeability transition and by stabilizing ΔΨm (Zamzami et al., J. Exp. Med. 183:1533–44 (1996)). In the absence of Bcl-2, apoptogenic factors, such as cytochrome c and apoptosis inducing factor (AIF), are released from mitochondria in response to apoptotic triggers (Susin et al., J. Exp. Med. 184:1331–41 (1996); Kluck et al., Science 275:1132–36 (1997)). This release in turn leads to sequential caspase activation and results in nuclear and membrane changes associated with apoptosis.
Bcl-2 family members display a distinct tissue-specific expression. In adult human liver, Bcl-2 expression is confined to bile duct cells (Charlotte et al., Am. J. Pathol. 144:460–65 (1994)) and is absent in both normal and malignant hepatocytes. In contrast, expression of Bcl-xL RNA and protein can be detected in adult quiescent hepatocytes and increases by 4 to 5 fold during the G1 phase of regenerating hepatocytes (Tzung et al., Am. J. Pathol. 150:1985–95 (1997)). Increased Bcl-xL expression is also observed in hepatoma cell lines, such as HepG2.
Some diseases are believed to be related to the down-regulation of apoptosis in the affected cells. For example, neoplasias may result, at least in part, from an apoptosis-resistant state in which cell proliferation signals inappropriately exceed cell death signals. Furthermore, some DNA viruses, such as Epstein-Barr virus, African swine fever virus and adenovirus, parasitize the host cellular machinery to drive their own replication and at the same time modulate apoptosis to repress cell death and allow the target cell to reproduce the virus. Moreover, certain diseases, such as lymphoproliferative conditions, cancer (including drug resistant cancer), arthritis, inflammation, autoimmune diseases, and the like, may result from a down regulation of cell death signals. In such diseases, it would be desirable to promote apoptotic mechanisms.
Most cancer chemotherapeutic agents that are currently available target cellular DNA and induce apoptosis in tumor cells (Fisher et al., Cell 78:539–42 (1994)). A decreased sensitivity to apoptosis induction has emerged as an important mode of drug resistance. In particular, over-expression of Bcl-2 and Bcl-xL confers resistance to multiple chemotherapeutic agents, including alkylating agents, antimetabolites, topoisomerase inhibitors, microtubule inhibitors and anti-tumor antibiotics, and may constitute a mechanism of clinical chemoresistance in certain tumors (Minn et al., Blood 86:1903–10 (1995); Decaudin et al., Cancer Res. 57:62–67 (1997)).
Neither Bcl-2 nor Bcl-xL, however, protects cells from every apoptotic inducer. For example, over-expression of Bcl-2 offers little protection against Thy-1-induced thymocyte death and Fas-induced apoptosis (Hueber et al., J. Exp. Med. 179:785–96 (1994); Memon et al., J. Immunol. 15:4644–52 (1995)). At the mitochondrial level, Bcl-2 over-expressed in the outer mitochondrial membrane inhibits PT pore induction by t-butyl-hydroperoxide, protonophore and atractyloside, but not by calcium ions, diamide or caspase 1 (Zamzami et al., J. Exp. Med. 183:1533–44 (1996); Susin et al., J. Exp. Med. 186:25–37 (1997)). Thus, one class of mitochondrially-active agents may directly affect the mitochondrial apoptosis machinery while bypassing the site of Bcl-2 function and the protection offered by Bcl-2 family members. An agent of this type may potentially be useful in overcoming the multi-drug resistance imparted by Bcl-2 or Bcl-xL and are of great need in the art.
The antimycins constitute another class of mitochondrially-active agents. The antimycins generally comprise a N-formylamino salicylate moiety linked to a dilactone ring through an amide bond. The antimycins differ in the hydrophobic R groups attached to the dilactone ring opposite the amide bond. (See, e.g., Rieske, Pharm. Ther. 11:415–20 (1980).) For example, antimycin A1 has a hexyl group at the 2 position of the dilactone ring while antimycin A3 has a butyl group at that position.) Extensive literature has been published on the structure-activity relationship of the antimycins and their inhibition of cytochrome bc1 (Miyoshi et al., Biochim. Biophys. Acta 1229:149–54 (1995); Tokutake et al., Biochim. Biophys. Acta 1142:262–68 (1993); Tokutake et al., Biochim. Biophys. Acta 1185:271–78 (1994)). The published structure of cytochrome bc1 complex with bound antimycin A1 reveals that antimycin A1 occupies a position in the Qi ubiquinone binding site on cytochrome b (Xia et al., Proc. Nat. Acad. Sci. USA 94:11399–404 (1997)). The antimycins generally inhibit mitochondrial respiration, which suggests that the differences in the hydrophobic R groups on the dilactone ring are not critical for cytochrome b binding. Mutagenesis and structure-activity studies of antimycin A demonstrate that the cytochrome bc1-inhibitory activity is highly dependent on the N-formylamino salicylic acid moiety (Tokutake et al. (1994), supra). Methylation of the phenolic hydroxyl or modification of the N-formylamino group both significantly reduce the ability of antimycin A to bind to and inhibit cytochrome bc1. Methylation of the phenolic hydroxyl diminishes inhibitory activity by 2.5 logs. Substitution of the formylamino group with acetylamino and propylamino groups at the 3-position reduce cytochrome bc1 activity by 1.2 and 2.4 logs, respectively. Thus, the N-formylamino salicylate moiety is generally understood to be important for binding of the antimycins to cytochrome b.
Two antimycins, antimycin A1 and A3, have recently been discovered to inhibit the activity of the anti-apoptotic Bcl-2 family member proteins, Bcl-2 or Bcl-xL. Thus, these molecules potentially useful compounds for the medical profession and patients suffering from proliferative disease and other diseases where apoptosis is inappropriately regulated. The antimycins are toxic, however, because they also inhibit mitochondrial respiration. There is a critical need, therefore, for derivatives of the antimycins that are effective in inducing apoptosis in cells where apoptosis is inappropriately regulated while exhibiting reduced inhibition of mitochondrial respiration.